Each year in the U.S., more than 23 000 people die as a result of antibiotic resistant infections (CDC 2014). To combat the growing spread of resistances to conventional antibiotics, new antibiotic development will be essential. Gene regulation by small non-coding RNAs (sRNAs) that act by base-pairing with mRNAs is an ideal target for such antibiotics, since sRNAs control cell growth, adaptability, and virulence processes in a wide variety of bacteria, including Enterobacteriaceae and Gram-positive pathogens. The long-term goal is to identify new antibiotic targets by understanding the mechanisms and proteins involved in sRNA-mediated gene regulation. Preliminary results indicate that the protein RNase PH has a previously unrealized role in regulating sRNAs. The objective of this research proposal is to determine the mechanisms by which RNase PH interacts with sRNAs to protect them from degradation, using Escherichia coli as a model system. This project will test the hypothesis that RNase PH selectively regulates sRNA degradation by interacting with sRNA 3' terminal regions to block binding and degradation by other RNases. Aim I will test the hypothesis that RNase PH protects both free and mRNA-bound sRNAs from degradation by preventing the binding of competing RNases. The effect of RNase PH on the stability of free and mRNA-bound sRNAs will be examined. Then the ability of RNase PH to protect against sRNA degradation by specific RNases will be tested, and its global impact on sRNA abundance and cellular fitness will also be examined. Aim II will test the hypothesis that RNase PH binds, but does not degrade, the Rho-independent terminators at the 3' termini of the sRNAs it protects. The sRNAs that directly bind to RNase PH will be isolated and sequenced to assess their integrity. The contributions of different sRNA features to stabilization by RNase PH will be examined through chimeric sRNA constructs and stability assays, and the sRNA-binding residues of RNase PH will be identified through mutagenesis, genetic screening, and stability assays. At its conclusion, this project is expected to (Aim I) demonstrate the scope and role of sRNA stabilization by RNase PH, as well as (Aim II) provide a mechanistic understanding of the features of sRNAs and RNase PH that enable this protection to occur. Understanding this novel role of RNase PH will inform the future development of new therapeutics effective against sRNA- mediated gene regulation.